Phosphoenolpyruvate carboxykinase (PEPCK) an early, key reaction in glucose synthesis in gluconeogenic cells. In most species, including man, two different forms of the enzyme are found, one in the mitochondria and one in the cytosol. The amount of PEPCK present in rat liver varies strikingly and rapidly in response to various endocrine and nutritional stimuli. However, rat tissues contain essentially only the cytosolic form of the enzyme. Up until the proposed studies there have been essentially no comparisons of the cytosolic and mitochondrial forms of PEPCK from the same tissue. Hence, little knowledge is available concerning the comparative structure of the two forms or the physiological and regulatory properties of the mitochondrial form of PEPCK. We have isolated both types of the enzyme in pure form from monkey liver and prepared antibodies against both antigens. The results indicate that the two forms are similar or identical in size and activity but can be separated electrophoretically and do not cross-react immunologically. The plans are to compare the structures of the two isozymes by amino acid analyses, end group determinations and peptide mapping to establish the degree of homology. The possibility that the catalytically active portions of the enzyme may be similar will be studied, initially by kinetic mapping of the PEP site through use of a series of PEP analogs which have been demonstrated to be alternate substrates or inhibitors. The regulation of the mitochondrial isozyme will be studied in guinea pigs by comparing the rates of synthesis and degration of the two isozymes in fed, fasted and diabetic animals.